Jun-Li Liu

Possible Roles of Reg Family Proteins in Pancreatic Islet Cell Growth

Reg proteins constitute a conserved family in human and rodents; their production in the pancreas (including the islets of Langerhans) is induced upon beta-cell damage. While some members of the family (Reg1 and islet neogenesis-associated protein, i.e. INGAP) have been implicated in beta-cell replication and/or neogenesis, including from in vivo studies using transgenic and knockout mice; the roles of the other five members have yet to be characterized. Among them, Reg2 was recently proposed to serve as an autoantigen on beta-cells that elicits T-cell attack in type 1 diabetes mellitus. Elucidation of their actions and identification of their molecular targets should provide insight into the biology of these proteins and lead to the design and development of novel strategies aimed at promoting the survival and function of the pancreatic islets. As the current terminology used for mammalian Reg genes/proteins is very confusing, we also proposed a uniformed classification in human and rodents through sequence alignments.

Overexpression of Reg3α increases cell growth and the levels of cyclin D1 and CDK4 in insulinoma cells

Regenerating gene (Reg) family protein Reg3α is normally expressed in pancreatic acinar and endocrine cells. In order to explore its effect on islet β-cell replication, insulinoma MIN6 cells were stably transfected with murine Reg3α cDNA. Determined using real-time PCR and Western blots, the levels of Reg3α mRNA and protein in Reg3α-transfected clones were increased 10- and 6-fold, respectively. Western blots also revealed that the protein was released into the culture medium, consistent with an endocrine effect. In MTT cell proliferation assay, Reg3α-overexpressing cells exhibited a 2-fold increase in the rate of cell growth. In order to investigate the intracellular mechanism, we studied cell cycle regulatory proteins. In Reg3α-expressing cells, we detected 2.2- and 2.5-fold increased levels of cyclin D1 and CDK4, respectively, which paralleled a 1.8-fold increase in the rate of Akt phosphorylation. It is established that β-cell replication is associated with increased cyclin D1 and CDK4 levels; deficiency in CDK4 or cyclin D2 results in reduced β-cell mass and diabetes. Our results suggest that Reg3α stimulates β-cell replication, by activating Akt kinase and increasing the levels of cyclin D1/CDK4.

Reg2 protects mouse insulinoma cells from streptozotocin-induced mitochondrial disruption and apoptosis

We reported previously that pancreas-specific ablation of IGF-I in mice induced an increased expression of regenerating family proteins Reg2 and Reg3β in the pancreas and protected them from streptozotocin (Stz)-induced β-cell damage. We, therefore, assessed the effect of ectopically introduced Reg2 on Stz-induced apoptosis in MIN6 mouse insulinoma cells and report here that Reg2 protects MIN6 cells from Stz-induced apoptosis by attenuating its ability to disrupt mitochondrial membrane integrity, activate caspase-3 and promote poly-ADP ribose polymerase cleavage, and induce apoptosis. These changes correlated with suppression of c-jun N-terminal kinase (JNK) phosphorylation by Stz. Reg2 inhibited Stz-induced proapoptotic events as well as the inactivation of JNK. Inclusion of chemical inhibitor of JNK to Reg2 expressing cells rendered them sensitive to Stz. These data demonstrate that Reg2 protects insulin-producing cells against Stz-induced apoptosis by interfering with its cytotoxic signaling upstream of the intrinsic proapoptotic events by preventing its ability to inactivate JNK.

IGF-I stimulates CCN5/WISP2 gene expression in pancreatic β-cells, which promotes cell proliferation and survival against streptozotocin.

IGF-I is normally produced from hepatocytes and other sources, stimulates protein synthesis, cell survival, and proliferation through receptor-mediated activation of phosphatidylinositol 3-kinase and MAPK, and targets specific molecules within the pancreatic islet cells. The current study was designed to identify novel targets that may mediate its pro-islet actions. Whole-genome cDNA microarray analysis in IGF-I-overexpressing islets identified 82 genes specifically up- or down-regulated. Prominent among them was CCN5/WISP2 whose expression was increased 3- and 2-fold at the mRNA and protein levels. Dual-labeled immunofluorescence revealed that CCN5 expression was low in the β-cells of wild-type islets but was significantly induced in response to IGF-I overexpression. In vitro treatment of mouse islets with IGF-I increased both CCN5 mRNA and protein levels significantly. To define the role of CCN5 in islet cell biology, we stably overexpressed its cDNA in insulinoma MIN6 cells and detected a 2-fold increase in the proliferation of MIN6-CCN5 compared with that in control cells, which correlated with significant elevations in the levels of cyclin D1 and the phosphorylation of Akt and Erk2. Moreover, MIN6-CCN5 cells were found to be resistant to streptozotocin-induced cell death. Using confocal microscopy and subcellular fractionation, we found that overexpressed CCN5 exhibited cytoplasmic accumulation upon stimulation by high glucose. Our results indicate that CCN5, which is minimally expressed in islet β-cells, is strongly and directly induced by IGF-I. CCN5 overexpression stimulates the proliferation of insulinoma cells, activates Akt kinase, and inhibits streptozotocin-induced apoptosis, suggesting that increased CCN5 expression contributes to IGF-I-stimulated islet cell growth and/or survival.

Intestinal adaptation and Reg gene expression induced by antidiabetic duodenal-jejunal bypass surgery in Zucker fatty rats

The antidiabetic mechanism of bariatric surgery includes specific changes in the secretion of incretins. To identify additional players originating from the gut, we evaluated the effects of duodenal-jejunal bypass (DJB) in morbidly obese Zucker fatty rats. A fast relief of hyperglycemia and hyperinsulinemia was achieved even before a significant weight loss occurred. Fourteen days after DJB, we characterized the changes in intestinal histochemistry in the bypassed duodenum and shortcut jejunum that was reanastomosed directly to the starting point of the duodenum and compared with the corresponding regions of sham-operated rats. The bypassed duodenum exhibited mucosal atrophy and apoptosis and decreased proliferative renewal. In shortcut jejunum, DJB resulted in 40% significantly enlarged intestinal circumference and increased epithelial proliferation, especially in putative transit-amplifying (TA) cells and the crypt. Because Reg family proteins promote cell growth and survival, we explored their expression in the intestine. With the use of immunohistochemistry, Reg1, -3α, and -3β were normally expressed in intestinal mucosa. After DJB, the level of Reg1 protein was reduced, whereas Reg3α and -3β were not changed in bypassed duodenum. Downstream in shortcut jejunum, the levels of Reg1 and -3β were greatly induced and especially concentrated in the putative TA cells. Our results revealed significant changes in the integrity and proliferation of the intestinal mucosa as a consequence of DJB, and in cell- and isoform-specific expression of Reg proteins within the replicating mucosal epithelium, and provide evidence indicating that the activation of Reg proteins may contribute to intestinal compensation against increased load and/or to improving insulin sensitivity.

A general IGF-I overexpression effectively rescued somatic growth and bone deficiency in mice caused by growth hormone receptor knockout.

Both growth hormone and insulin-like growth factor (IGF)-I are essential for postnatal somatic growth, while exerting distinct effects on energy homeostasis. Although growth hormone controls IGF-I production, whether IGF-I was the exclusive mediator of its growth promotion is still debated. In order to further explore their in vivo interactions in somatic growth as well as in energy homeostasis, we have crossed mutant (MT-IGF) transgenic mice onto the GHR − / − background. As expected, GHR gene deficiency caused growth retardation, including significant decreases in lumbar, femur and total body lengths, as well as decreased bone area, mineral content and mineral density. IGF-I overexpression alone in MT-IGF mice increased the weight, with no significant change in bone mineralization or longitudinal growth. Compared to GHR − / − littermates, overexpressed IGF-I in bitransgenic mice (GHR − / − and MT-IGF positive) exhibited fully restored body weight, lumbar (but not femur) and total body lengths, and normalized overall bone area, mineral content and density. On the other hand, there were significant changes in fasting glucose level, glucose tolerance, lean/fat masses and even adipose histology as a result of the transgenic/knockout double-crossing. IGF-I overexpression normalized glucose tolerance in GHR − / − mice. Intriguingly, on GHR+/ − background of partial growth hormone insensitivity, overexpression of IGF-I caused a significant weight gain. Our results thus establish that the growth defect and bone deficiency caused by lack of growth hormone signaling can be effectively restored by increasing IGF-I production in vivo.

Coordinated age-dependent and pancreatic-specific expression of mouse Reg2Reg3α, and Reg3β genes

Reg family proteins such as Reg1 and islet neogenesis-associated protein (INGAP) have long been implicated in the growth and/or neogenesis of pancreatic islet cells. Recent reports further suggest similar roles to be played by new members such as Reg2, Reg3α, and Reg3β. We have studied their age-, isoform-, and tissue-specific expressions. RNA and protein were isolated from C57BL/6 mice aged 7, 30, and 90 days. Using real-time polymerase chain reaction, the levels of Reg gene expression in the pancreas were 20–600-fold higher than that in other tissues (≫duodenum>stomach>liver); gene expression of Reg2, Reg3α, and Reg3β was age dependent as it was hardly detectable at day 7, increased drastically at day 30, and significantly decreased at day 90; the levels of pancreatic proteins displayed similar age-dependent variations. Using dual-labeled immunofluorescence, Reg2, Reg3α, and Reg3β were abundantly expressed in most acinar cells of the pancreas, in contrast to INGAP which exhibited stepwise increases from day 7 to day 90 and colocalized with the α-cells. These new Reg genes were mainly expressed in the pancreas, with clear age-dependent and isoform-specific patterns.

Pancreatic acinar-specific overexpression of Reg2 gene offered no protection against either experimental diabetes or pancreatitis in mice

Reg proteins are normally expressed in pancreatic acinar cells, and the level of several of these proteins was significantly induced upon damage to the endocrine or exocrine pancreas. It has been established that Reg1 and pancreatic islet neogenesis-associated protein [INGAP, Reg3delta] promote the growth or regeneration of the endocrine islet cells. Recent reports suggest that Reg2 is an autoantigen normally expressed in islet beta-cells. Reg2 overexpression in vitro offered protection to insulinoma cells. Overexpressed Reg3alpha increased cyclin D1 and CDK4 levels and the rate of proliferation in insulinoma cells. Acinar-specific overexpression of INGAP increased beta-cell mass and protected the animals from streptozotocin-induced diabetes. Moreover, Reg2 gene expression was induced during pancreatitis. We hypothesized that Reg2 is a secreted protein that promotes the growth, survival, and/or regeneration of pancreatic endocrine and exocrine cells. To test its effectiveness, we used elastase-1 promoter (Ela-Reg2) to develop an acinar cell-specific overexpression of the Reg2 gene. Western blot analysis, real-time PCR, and immunohistochemistry revealed barely detectable levels of endogenous Reg2 in the pancreas of normal wild-type mice and increased Reg2 levels in the pancreas of Ela-Reg2 mice that were similar to or higher than Reg2 levels induced in experimental diabetes or pancreatitis. Compared with wild-type littermates, growth, blood glucose and insulin levels, and glucose tolerance were normal in Ela-Reg2 mice; pancreatic histology revealed no change in endocrine or exocrine tissues. Acinar-specific overexpression of the Reg2 gene offered no protection against streptozotocin-induced beta-cell damage and diabetes, in hyperglycemia and weight loss, and no advantage in restoring glucose homeostasis and islet function within 3 mo. Furthermore, serum amylase level and pancreatic histochemistry showed that Reg2 overexpression did not protect acinar cells against caerulein-induced acute pancreatitis. In contrast to INGAP or Reg3beta, exocrine overexpression of Reg2 offered no protection to the endocrine or exocrine pancreas, indicating clear subtype specificities of the Reg family of proteins.

Parp1 deficient mice are protected from streptozotocin-induced diabetes but not caerulein-induced pancreatitis, independent of the induction of Reg family genes.

Poly(ADP-ribose) polymerase (Parp) 1 is a key regulator of cell death, its inhibition prevented streptozotocin-induced diabetes and attenuated caerulein-induced acute pancreatitis. Reg family proteins are significantly induced by Parp1 inhibitor, experimental diabetes and/or acute pancreatitis. We propose that Reg proteins are involved in the protection of pancreatic cells by Parp1 inhibition. To test this possibility, Parp1-/- and wild-type mice were injected with streptozotocin to induce diabetes. Separately, acute pancreatitis was induced with repeated injections of caerulein. Upon streptozotocin administration, Parp1-/- mice displayed much decreased hyperglycemia and preserved serum insulin level. The treatment induced similar levels of Reg1, -2, -3α and -3β genes in the pancreas of both wild-type and Parp1-/- mice, suggesting that the upregulation of Reg family genes during streptozotocin-induced diabetes was independent of Parp1 ablation. In caerulein-induced pancreatitis, unlike being reported, Parp1 knockout caused no relief on the severity of pancreatitis; the upregulation of pancreatic Reg1, -2, -3α and -3β genes upon caerulein was unaffected by Parp1 deletion. Our results reconfirmed the protective effect of Parp1 gene deletion on islet β-cells but questioned its effect on the acinar cells. In either case, the significant induction of Reg family genes seemed independent of Parp1-mediated cell death.

Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells

Murine regenerating (mReg) genes have been implicated in preserving islet cell biology. Expanding on our previous work showing that overexpression of mReg2 protects MIN6 insulinoma cells against streptozotocin‐induced apoptosis, we now demonstrate that mReg2 induces glucose‐regulated peptide 78 (GRP78) expression via the Akt–mTORC1 axis and protects MIN6 cells against ER stress induced by thapsigargin and glucolipotoxicity. Activation of mTORC1 activity results from both mReg2‐induced increased mTOR phosphorylation as well as increased expression of Raptor and GβL. Inhibition of Akt and mTORC1 blunted the ability of mReg2 to induce GRP78 and attenuate unfolded protein response (UPR). Knockdown of GRP78 sensitized the cells overexpressing mReg2 to UPR without affecting its ability to activate Akt–mTORC1 signaling. Induced expression of mReg2 may protect insulin producing cells from ER stress in diabetes.